1Department of Surgery, University of Pittsburgh, Pittsburgh, PA. 2Department of Sports Medicine and Nutrition, University of Pittsburgh, Pittsburgh, PA. 3Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA. 4Department of Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA. 5Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI. 6Center for Inflammation and Regenerative Modeling, McGowan Institute for Regenerative Medicine, Pittsburgh, PA.

Abstract

OBJECTIVE:

Blunt trauma patients may present with similar demographics and injury severity yet differ with regard to survival. We hypothesized that this divergence was due to different trajectories of systemic inflammation and utilized computational analyses to define these differences.

DESIGN:

Retrospective clinical study and experimental study in mice.

SETTING:

Level 1 trauma center and experimental laboratory.

PATIENTS:

From a cohort of 493 victims of blunt trauma, we conducted a pairwise, retrospective, case-control study of patients who survived over 24 hours but ultimately died (nonsurvivors; n = 19) and patients who, after ICU admission, went on to be discharged(survivors; n = 19).

INTERVENTIONS:

None in patients. Neutralizing anti-interleukin-17A antibody in mice.

MEASUREMENTS AND MAIN RESULTS:

Data on systemic inflammatory mediators assessed within the first 24 hours and over 7 days were analyzed with computational modeling to infer dynamic networks of inflammation. Network density among inflammatory mediators in nonsurvivors increased in parallel with organ dysfunction scores over 7 days, suggesting the presence of early, self-sustaining, pathologic inflammation involving high-mobility group protein B1, interleukin-23, and the Th17 pathway. Survivors demonstrated a pattern commensurate with a self-resolving, predominantly lymphoid response, including higher levels of the reparative cytokine interleukin-22. Mice subjected to trauma/hemorrhage exhibited reduced organ damage when treated with anti-interleukin-17A.

Trauma patients were recruited following IRB approval and informed consent. (A) A daily Marshall MOD score analysis for organ failure exhibited a slight decrease in Marshall MOD score over 7d in survivors. The non-survivors exhibited later-onset and increasing multiple organ dysfunction, reaching a state of sustained multiple organ failure (MOD score ≥ 5) by Day 5, significant (P<0.05) by Two-Way ANOVA over 7d. Plasma was obtained at multiple time points and analyzed for the presence of 27 inflammatory mediators in highly-matched sub-cohorts of survivors and non-survivors, followed by Dynamic Network Analysis (DyNA) as described in the Materials and Methods. (B) The time-evolution of networks in non-survivors (framed in red) and survivors (framed in green). In silico inference of inflammatory network complexity suggests a bifurcation of low-level versus self-amplifying inflammation in survivors and non-survivors, respectively. Notably, in silico-defined trajectories of inflammation mimic the clinical trajectories of organ dysfunction over 7d in both survivors and non-survivors.

Trauma patients were recruited following IRB approval and informed consent. Plasma was obtained at multiple time points and analyzed for the presence of 27 inflammatory mediators in highly-matched sub-cohorts of survivors and non-survivors, followed by Dynamic Network Analysis (DyNA) as described in the Materials and Methods. (A) Inferred dynamic networks in the survivors group predominantly involved lymphoid-associated cytokines. Strongly connected networks of interaction between IL-17A, IL 4, and sIL-2Rα; IL-22 and IL-23; and between IL-5 and IL-13, were established early on and persisted over the 7d course in survivors. (B) Non-survivors were characterized by networks of both innate mediators and lymphoid mediators that evolved into much larger, innate-dominant networks by 72h. IL-17 in the figure refers to IL-17A. All original DyNA outputs are included in .

Trauma patients were recruited following IRB approval and informed consent. Plasma was obtained at multiple time points and analyzed for the presence of 27 inflammatory mediators in highly-matched sub-cohorts of survivors and non-survivors, and the data were subjected to Bootstrapped Spearman’s rank correlation as described in the Materials and Methods. This analysis suggests a positive correlation between IL-17A and GM-CSF and a negative correlation between IL-17A and IL-10, consistent with a pathogenic Th17 phenotype. No significant correlation was found among these mediators in the survivors sub-cohort. The shaded area in pink represents the 95% confidence interval.

Mice (n=6 in all groups) were subjected to an experimental hemorrhagic shock and trauma model (HS/T) as described in the Materials and Methods. (A) Time course of circulating IL-17A/F in mice subjected to HS/T. *: P<0.05 vs. baseline which quickly diminished by 12h and 24h time points. (B and C) Neutralization of IL-17A/F by an anti-IL-17 monoclonal antibody at 6h resulted in significantly decreased plasma IL-17A/F when compared to the control group by Mann-Whitney Rank Sum test (panel B; P=0.002) and a marked decrease in alanine aminotransferase (ALT), a marker of liver dysfunction, when compared to the control group by t-test (panel C; P<0.001).

A hypothetical “inflammatory switch state”, involving innate and lymphoid mediators early on following trauma. In this paradigm, the magnitude of a pro- and anti-inflammatory response remains a critical aspect in determining clinical outcomes. (A) A well-balanced lymphoid signature following traumatic injury involves Treg, Th2, and nonpathogenic Th17 (Th17NP) cells. In contrast, the early activation of a pathogenic Th17 (Th17P) phenotype is triggered when the balance between lymphocytes is tipped leading to an uncontrolled inflammatory cascade – much like the balance of lymphocytes is tipped in those predisposed to develop chronic inflammatory diseases. (B) The severity of pro- and anti-inflammatory mediator involvement [represented horizontally in the x-axis] is either attenuated by a balanced lymphoid response or, conversely, exacerbated by a self-sustaining, predominantly innate-mediated response. Thus, this diagram represents the two major outcomes of these contrary inflammatory patterns: (1) a well-balanced lymphoid response leads to injury resolution and (2) a loss of lymphocyte regulation engages self-sustaining inflammation that impedes injury resolution.